Qingfei Formula Protects against Human Respiratory Syncytial Virus-induced Lung Inflammatory Injury by Regulating the MAPK Signaling Pathway

Objective Qingfei formula (QF) is an empirical formula that shows good clinical efficacy in treating human respiratory syncytial virus pneumonia (RSVP). However, the underlying mechanism remains unclear. This study explores the possible pharmacological actions of QF in RSVP treatment. Methods We used a network pharmacology approach to identify the active ingredients of QF, forecast possible therapeutic targets, and analyze biological processes and pathways. Molecular docking simulation was used to evaluate the binding capability of active ingredients and therapeutic targets. Finally, in vivo experiments confirmed the reliability of network pharmacology-based prediction of underlying mechanisms. Results The study identified 92 potential therapeutic targets and corresponding 131 active ingredients. Enrichment analysis showed that QF downregulated the MAPK signaling pathway and suppressed the inflammatory injury to the lungs induced by the RSV virus. Molecular docking simulations demonstrated that the core active ingredients of QF could stably bind to genes associated with the MAPK signaling pathway. QF had a protective effect against pneumonia in RSV-infected mice. The QF group exhibited a significant reduction in the levels of inflammatory mediators, interleukin-6 (IL-6), interleukin-8 (CXCL8, IL-8), and P-STAT3, compared to the RSV-induced group. The QF group showed remarkably inhibited MAPK1+3(P-ERK1+2) and MAPK8(P-JNK) protein expression. Conclusion The current study showed that QF downregulated the MAPK signaling pathway, which inhibited pulmonary inflammation triggered by RSV infection. This study recommends the appropriate use of QF in the clinical management of RSVP.


INTRODUCTION
The human respiratory syncytial virus (RSV) belongs to the genus Pneumovirus of the family Paramyxoviridae.It is a single-stranded negative-sense virus.The viral RNA encodes 10 subgenomic mRNAs and 11 proteins, and the genome is contained in a nucleocapsid enveloped in a lipoprotein [1].The primary surface glycoprotein (G) enables virus attachment, and the fusion protein (F) mediates virus-cell fusion [2].RSV is a major contributor to pediatric hospitalizations due to RSV pneumonia (bronchiolitis or interstitial 1875-5402/24 pneumonia) in infants, young children, and the elderly.Moreover, RSV is a factor in the deterioration of health in adults [3].Intensive research on the treatment and control of RSV has been conducted, but no vaccinations or specific medicines are available.In view of the resultant lung tissue damage caused by RSV infection, it is very important to find a more suitable therapeutic strategy.
The Qingfei formula (QF) is a commonly used TCM prescription improvised from the classical prescription of Maxing Shigan decoction.QF has been used to treat viral pneumonia, especially human respiratory syncytial virus pneumonia (RSVP), for more than 40 years [4].It (Quanshen).QF was found to play a vital regulation role in the immune system and inflammation after RSV infection [4].However, the underlying mechanism is still unclear, and this lack of knowledge limits its therapeutic use.As a new area of pharmacology, network pharmacology provides new methods for elucidating the multiple mechanisms of the actions of drugs by exploring the disease targets [5].This study used network pharmacology and in vivo experimental verification to provide a preliminary determination of the mechanism of QF in RSVP treatment.Fig. (1) displays the flow chart of the study.

Fig. (1). The flow chart of this study. (A higher resolution / colour version of this figure is available in the electronic copy of the article).
The interaction between QF targets and RSVP targets was examined using the R software to identify potential therapeutic targets [9].

GO and KEGG Analyses
We used the R package clusterProfiler for Gene Ontology (GO) function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis to demonstrate the role of potential therapeutic targets in gene function and signaling pathways [10].Following the completion of the GO function enrichment study, which comprised biological process (BP), cellular component (CC), and molecular function (MF) items, the top ten pertinent GO enrichment items were displayed as bubble plots.The top twenty pertinent KEGG pathways were presented as bubble plots.

Construction of Ingredient-Target Network
Our study thoroughly examined the molecular mechanism of QF in treating RSVP by creating an active ingredient-potential therapeutic target network diagram using Cytoscape 3.6.0software [11].The core nodes of the network were analyzed using the DegreeFilter plugin.The top five pharmaceutical ingredients (according to degree value) were considered the core active ingredients of QF.

Construction of Protein-protein Interaction Network
The STRING database (https://string-db.org)seeks to assemble and combine all publicly accessible sources of protein-protein interaction (PPI) knowledge to develop a comprehensive network [12].To assess the interactions among the potential therapeutic targets, we uploaded the potential therapeutic targets and created a PPI network graph (interaction score = 0.7).Then, we loaded the PPI data into the Cytoscape 3.6.0software to visualize results and used the Cy-toNCA plugin to determine the core genes.

Molecular Docking Simulation
Molecular docking was used to assess the binding between core genes and core active ingredients.Molecular docking simulations were performed following established protocols using AutoDock 4.2 and AutoDock Vina software [13].

Protocol of Animal Experiments In Vivo
Fifty specific pathogen-free grade BALB/c mice (20.0 ± 2.0 g; 6 -8 weeks) were purchased from Jiangsu Qin-glongshan Co., Ltd.(Jiangsu, China).The mice were randomly divided into 5 groups (n=10): the control group, the RSV-induced group (Model), the QF low-dose (QF-LD) group (0.2 mL/d), the QF high-dose (QF-HD) group (0.6 mL/d), and the ribavirin control group (Riba) (46 mg/kg/d).All mice, excluding those in the control group, were given an intranasal injection of the RSV virus (1.4 × 107 plaqueforming units) under a light anesthetic (isoflurane).Mice in the control group received the same volume of sodium chloride solution.The treatment groups received oral medications 48 hours after infection.Sodium chloride solution was administered to the control and model groups in equal amounts.The dosage continued for three days, and the mice were observed daily for health status.Mice were sacrificed 5 days after infection to collect pertinent samples [14].All animal experiments were approved by the Institutional Animal Care and Use Committee of the Laboratory Animal Services Center at Nanjing University of Chinese Medicine (approval ID: SYXK (Su) 2018-0049) and performed according to the relevant guidelines and regulations.

Pathological and Immunohistochemical Examination
After preservation with a 4% paraformaldehyde solution for 24 hours, mouse lung tissue was dehydrated.The mouse samples were divided into 3-mm thick sections after paraffin embedding.Hematoxylin and eosin (HE) staining was applied to the slices before microscopic examination in a double-blind fashion.The average of five fields in each section, rated from 0 to 3 (normal, mild, moderate, and severe), was used to reflect the degree of pathological alterations [15,16].Additional tissue samples were used from each group for immunohistochemistry tests, followed by P-STAT3 expression analysis.The expression of the favorable outcomes was cytoplasmic brown staining.The study used Image-Pro Plus 6.0 software to calculate the average integrated optical density (IOD) [17].

Immunofluorescence Assay
Lung tissues were deparaffinized and dehydrated.Following antigen retrieval, lung slices were processed for immunofluorescence.The fixed tissue samples were placed on the cover glass, blocked with donkey serum (Solarbio, Beijing, China), and probed with RSV-antibody [18].The fixed tissue sections were rinsed thrice with PBS before incubation with the secondary antibody at 37°C for 50 min while keeping out of the light.The cell nuclei were stained with 4′,6diamidino-2-phenylindole at 37°C for 10 min in the dark.Finally, an anti-fade mounting buffer was applied to the sectioned tissues for fluorescence microscopy (Olympus).

Western Blot Analysis
Western blot analysis was performed following the standard procedure [19].Equal amounts of protein were separated using 10% SDS PAGE and then transferred to a PVDF membrane (Merck Millipore, IRL).The membrane was treated with the primary antibody overnight at 4°C, followed by an hour-long incubation with the corresponding secondary antibody at room temperature to block nonspecific binding sites.Antigen-antibody complexes were discovered using the ECL reagent (Yeasen, Shanghai, China).
Sun et al.

Statistical Analysis
GraphPad Prism 5.0 was used for the statistical analysis.All data have been reported as mean ± standard error and gathered from at least three different experiments.A comparison of the means of several groups was made using a one-way analysis of variance test.The levels of significance were specified at 0.05 and 0.01.

Identification of Potential Therapeutic Targets
We combined and deduplicated the search results after filtering them based on the herbal names and screening conditions.The following were the active ingredients in QF, according to the results: 23 species of Mahuang, 1 species of Shigao, 19 species of Xingren, 32 species of Sangbaipi, 12 species of Tinglizi, 24 species of Qianhu, 10 species of Huzhang, 6 species of Quanshen, 2 species of Jiangcan, and 65 species of Danshen.After deleting 27 duplicate ingredients, we obtained 167 active ingredients.Five active ingredients in QF were obtained through a literature search.Further search for the active ingredient targets yielded a total of 247 targets.
We searched the GeneCards, DisGeNET, and OMIM databases and obtained 685 RSVP targets, 2 RSVP targets, and 71 RSVP targets.A total of 740 RSVP targets were obtained after integrating the outcomes of the three databases and eliminating the duplicates.The 247 QF targets and 740 RSVP targets were then overlapped to yield potential therapeutic targets.Ultimately, interaction analysis produced 92 potential therapeutic targets (Fig. 2A and Table 1).Then, potential therapeutic targets were classified according to their biochemical criteria, as shown in Fig. (2B).These 92 potential therapeutic targets mainly included gene-specific transcriptional regulators and protein-modifying enzymes.

Biological Function Analysis of Potential Therapeutic Targets
GO function enrichment analysis showed the top ten GO items for BP, CC, and MF, and KEGG pathway enrichment analysis identified 20 KEGG pathways.GO functional enrichment results primarily identified a reaction to lipopolysaccharides, molecules of bacterial origin, and oxidative stress as BP items of potential therapeutic targets.Potential therapeutic targets for CC items mainly comprised membrane raft, membrane microdomain, and membrane region.Potential therapeutic targets for MF items comprised cytokine receptor binding, receptor-ligand activity, and cytokine activity.KEGG results showed AGE-RAGE signaling route, the PI3K-Akt signaling pathway, and Kaposi's sarcomaassociated herpesvirus infection signaling pathway to be the key signaling pathways linked with potential therapeutic targets (Fig. 3A and 3B).

Ingredient-Target Network
The ingredient-target network, depicted in Fig. (4A), was built to clarify the interactions between them.It had 223 nodes (92 potential therapeutic targets and corresponding 131 active ingredients).The nodes had different colors, with blue representing potential therapeutic targets and light blue representing the active ingredients.The edges were used to indicate the correlation between the nodes.The network analysis revealed that the average degree value for the active ingredients was 9.08, suggesting that QF has several targets for treating RSVP.Notably, the network contained five active ingredients with degrees 25, and these five ingredients considered to be the core active ingredients of QF were quercetin (degree = 416), luteolin (degree = 96), kaempferol (degree = 68), beta-sitosterol (degree = 61), and tanshinone (degree = 30) (Fig. 4B).

PPI Network Analysis
For PPI network development and analysis, 92 potential therapeutic targets were loaded into the STRING database, and 813 edges reflecting the interaction between proteins (interaction score = 0.7) were produced by the network's 89 interacting targets (SOAT1, DUOX2, and GSTM1 were not involved in protein interaction) (Fig. 5A).Based on three key parameters, betweenness centrality (BC), closeness centrality (CC), and degree centrality (DC), the topological feature analysis of the PPI network chose targets above median values as the core genes of prospective therapeutic targets.The first screening's threshold values were BC = 26.8,CC = 0.5, and DC = 14, and 36 nodes and 386 edges were the final results.The key genes, including EGF, MAPK1 (ERK2), MAPK3 (ERK1), MAPK8 (JNK1), JUN, STAT3, IL-6, and CXCL8 (IL8), were discovered after four screenings (Fig. 5B).

Molecular Docking and Analysis
Molecular docking was performed to simulate the binding properties of various ingredients and key genes.The screening results mentioned above allowed for verifying EGF, MAPK1, MAPK3, MAPK8, JUN, STAT3, IL-6, and CXCL8.In Autodock, the 3D structure was imported and docked with various compounds, including quercetin, luteolin, kaempferol, beta-sitosterol, and tanshinone.

QF Mitigated RSV-induced Lung Injury and Inhibited Virus Replication
H&E staining was used to examine the histological alterations in the lung tissue.According to Fig. (7A), mice lung tissue in the control group showed distinct alveolar lobules and alveolar cavities without leakage or cell infiltration in the alveolar gaps or the interstitium.RSV infection resulted in severe pulmonary inflammation characterized by lung consolidation, thickening of the alveolar wall, and lymphocytic infiltration.Inflammatory cells also invaded the alveolar space and lung interstitium due to the RSV infection.Compared to the model group, the QF-treated groups showed significantly lower scores for pathological damage and lung injury (lung consolidation, thickening of the alveolar wall, and lymphocyte infiltration), with mild inflammatory cell infiltration and protein leakage in the alveolar cavity (Fig. 7C-E).
The severity of RSV infection is associated with the level of virus amplification.The relative expression of RSV-F genes was detected using QPCR to confirm RSV virus replication in the lung.As shown in Fig. (7F), RSV infection caused a marked increase in RSV-F mRNA levels in mouse lung tissue.Compared to the model group, the RSV-F mRNA levels in the QF-treated groups showed a significant dose-dependent reduction.We used an immunofluorescence technique to measure the virus levels in the mouse lung and track the challenge dosage of the virus (Fig. 7B).Quantitative analysis showed that the amount of virus in the QFtreated groups was much lower than in the model group (Fig. 7G).Overall, these findings showed that, in RSV-infected mice, QF efficiently reduced lung damage and prevented virus multiplication.

Effects of QF on Potential Targets
We used Western blotting to assess the expression of proteins involved in the MAPK pathway to confirm the outcomes of network pharmacology and investigate the significance of prospective therapeutic targets.Results showed that P-ERK(1+2) and P-JNK expression levels in the model group were significantly higher than in the control group, proving that RSV might activate the MAPK signaling pathway.Compared to the model group, QF and ribavirin significantly decreased P-ERK(1+2) and P-JNK levels after RSV infection (Fig. 8A-C).These findings imply that QF reduces RSV-induced inflammation by preventing MAPK signal activation.
We assessed the level of inflammation-related genes in the MAPK signaling pathway to confirm the abovementioned finding.We conducted immunohistochemistry tests on mouse lung tissues.The outcomes demonstrated that the RSV model mice lung tissue had higher levels of P-STAT3 protein expression than the control group.Compared to the model group, the protein expression of P-STAT3 in lung tissue was significantly lower in the QF-treated groups (Fig. 9A and B).We also assessed IL-6 protein concentrations in the lung.Compared to the model group, the protein expression of IL-6 in the QF-treated groups dramatically decreased (Fig. 9C and D).The same pattern was seen in IL-8 mRNA expression levels (Fig. 8D).

DISCUSSION
RSV-related lower respiratory tract infections are a substantial cause of death in young children, with over 200,000 occurrences per year worldwide [20].RSV infections repeat throughout adulthood because an organism does not acquire long-lasting immunity against the infection.RSV infection is also common in elderly and immunosuppressed people [21].A previous study on the elderly in the United States found that RSVP imposes a significant medical and economic burden on the elderly: 14,000 to 62,000 cases of RSV-related pneumonia hospitalization occur every year, with an estimated cost of $150 to $680 million [22].Despite significant morbidity, there are currently no efficient vaccinations.The US Food and Drug Administration has approved the antiviral medications ribavirin and palivizumab for treating severe RSV infections.However, the use of these medications is limited by the significant risk of toxicity associated with ribavirin and high palivizumab cost [23].
Given these limitations, Chinese herbal medicine applications need to be promoted.In a previous multicenter clinical study, QF exhibited remarkable therapeutic effects on viral pneumonia, especially RSVP [24].Specifically, compared to ribavirin, QF shortened the body temperature recovery time and significantly improved cough, copious sputum, and shortness of breath symptoms in patients [25].QF dramatically decreased the exudation of inflammatory mediators (IL-6, 8) in mice with RSV-induced viral pneumonia and restored the Th1/Th2 imbalance, according to in vivo investigations [26][27][28].
This study used network pharmacology to investigate the mechanism of action of QF in treating RSVP.Molecular docking was used concurrently to simulate the binding properties of core active ingredients and core genes.Finally, in vivo test outcomes were obtained.These findings will be a foundation for future studies on RSVP treatment with QF.
We used a network pharmacology technique to discover 131 active components and 92 possible therapeutic targets.The top five active ingredients, quercetin, luteolin, kaempferol, beta-sitosterol, and tanshinone, were the core active ingredients in QF with the strongest correlations, according to the degree value ranking in the active ingredientpotential therapeutic target network.Reports show that some of these active substances may directly combat pneumonia viruses by reducing inflammation.Quercetin dramatically decreased the lung inflammation and mortality caused by LPS in mice and prevented the release of serum necrosis factor, interleukin-1, and interleukin-6 [29].Luteolin and kaempferol are potential protective antagonists of acute lung injury in mice because they can suppress the activation of MAPK and NFκB pathways triggered by LPS [30,31].Tanshinone prevents inflammation and apoptosis and reduces acute lung injury caused by LPS in mice; β-sitosterol improves influenza symptoms.Virus-mediated recruitment of pathogenic cytotoxic T cells and immunological dysregulation can protect mice against deadly influenza A virus infection [32,33].These findings demonstrate the validity and viability of the network pharmacology approach to the search for active substances.Future studies should focus on the potential of active components to produce direct antiviral or anti-inflammatory effects.
Enrichment analysis of 92 prospective therapeutic targets showed that QF regulated the AGE-RAGE signaling pathway, the PI3K-Akt signaling system, and the herpesvirus infection linked to Kaposi sarcoma in vivo.According to the PPI network's topological feature analysis findings, QF could inhibit the MAPK signaling pathway to prevent the overexpression of inflammatory mediators in RSV-infected mice.The three main signaling routes (AGE-RAGE signaling pathway, PI3K-Akt signaling pathway, and Kaposi's sarcoma-associated herpesvirus infection) are strongly related to the MAPK signaling pathway [34][35][36].Thus, to support this theory, we conducted in vivo tests.
Histopathological sections revealed lung consolidation, thickening of the alveolar wall, and lymphocytic infiltration in lung tissues of the model group mice.PCR results showed a significant increase in the RSV virus mRNA levels of the model group mice.However, the QF groups (LD and HD) exhibited a significant improvement in lung tissue injury and RSV virus mRNA levels in the lung.The results of the immunofluorescence assay were similar to PCR results.We also investigated whether the anti-inflammatory effects of QEOL involve the MAPK signaling pathway in the lung.Reports suggested that the MAPK signaling pathway showed significant activation during RSV infection in human alveolar basal epithelial cells [37].Inflammation was found to be greatly influenced by the MAPK signaling pathway [38].The p38 MAPK, ERK, and JNK pathways are interconnect-ed MAPK pathways.Viral infection is one of the many intracellular and extracellular triggers that can activate these serine/threonine protein kinases.Several downstream transcription factors essential in controlling inflammation, including STAT-3, IL-6, and IL-8, are subsequently activated by p38 MAPK, JNK, and ERK [39][40][41][42][43][44].The findings have demonstrated a considerable increase in the expression of P-ERK1+2 and P-JNK proteins in the lung tissue of the model group mice, indicating the activation of the MAPK signaling pathway.Inflammatory factors, such as IL-6, IL-8, and P-STAT3, have been found to be overexpressed concurrently with the MAPK signaling pathway activation.Oral QF therapy prevented P-ERK1+2 and P-JNK protein overexpression and decreased IL-6, IL-8, and STAT3 levels.These results revealed that QF controlled the MAPK signaling pathway to prevent excessive inflammatory responses.The outcomes further confirmed the dependability of the network pharmacology prediction target.

CONCLUSION
The network pharmacological analysis demonstrated QF to be a complex preparation with multi-ingredient and multitarget properties, identifying 131 active components and 92 possible therapeutic targets linked to RSV virus infection.By controlling several targets, primarily those related to quercetin, luteolin, kaempferol, beta-sitosterol, and tanshinone, QF exerted therapeutic effects on RSVP.After the enrichment analysis of 92 potential therapeutic targets and topological feature analysis of the PPI network, we found that the MAPK signaling pathway plays an important role in the anti-inflammatory effects of QF in treating RSVP.Furthermore, molecular docking simulation confirmed genes related to the MAPK pathway, including EGF, MAPK1, MAPK3, MAPK8, JUN, STAT3, IL-6, and CXCL8, to exhibit good binding affinities with the corresponding active ingredients.According to the core gene screening results, we verified the effect of QF on RSV-infected mice and determined that QF exerts anti-inflammatory effects by downregulating the MAPK signaling pathway.In vivo experiment results further confirmed the reliability of network pharmacology to predict potential therapeutic targets.These findings offer a fresh approach to RSVP treatment and details on the mechanisms of QF.

ETHICS APPROVAL AND CONSENT TO PARTICI-PATE
This study was approved by the Animal Ethics Association of Nanjing University of Chinese Medicine, Nanjing, China (Approval no.SVXK(Su) 2018-0049).

HUMAN AND ANIMAL RIGHTS
All animal procedures were performed according to the guidelines for the ethical review of laboratory animal welfare at Najing University of Chinese Medicine.

CONSENT FOR PUBLICATION
Not applicable.

Fig. ( 2
Fig. (2).Prediction of the potential therapeutic targets.(A) Venn diagram of the potential therapeutic targets.(B) The classification of potential therapeutic targets according to their biochemical criteria.(A higher resolution / colour version of this figure is available in the electronic copy of the article).

Fig. ( 4
Fig. (4).(A) Active ingredient-potential therapeutic target network.(B) Five core ingredients in potential therapeutic target network (red represents ingredients; yellow and green represent targets).(A higher resolution / colour version of this figure is available in the electronic copy of the article).

Fig. ( 5 )
Fig. (5).Protein-protein interaction network analysis.(A) PPI network.(B) The screening process of core genes.(A higher resolution / colour version of this figure is available in the electronic copy of the article).
Fig. (6A) shows the energy values of the chemicals used in the docking results.Fig. (6B) depicts the molecular docking pattern of tanshinone with MAPK3.These low docking energy values suggested that the ingredients might stably bind to the genes.Indirect evidence from the data further validates the validity of the network pharmacology prediction target by demonstrating agreement between the molecular docking and network pharmacology screening results.

Fig. ( 6 ).
Fig. (6).Molecular docking and analysis.(A) Heat map of the docking energy value.(B) Molecular docking pattern of tanshinone with MAPK3.(A higher resolution / colour version of this figure is available in the electronic copy of the article).

Fig. ( 7 )
Fig. (7).QF mitigated RSV-induced pulmonary histopathological damage and inhibited virus replication.(A) Pathological changes in lung tissue induced by RSV.Scale bar, 100 μm.(B) RSV expression assessed via an immunofluorescence assay.Scale bar, 50 μm.(C-E) Lung injury scores according to the degree of lung damage.(F) RSV-F mRNA level.(G) RSV fluorescence intensity.Data are presented as mean ± standard error.Significance: ## P <0.01 vs. control group; # P <0.05 vs. control group; ** P <0.01 vs. model group; * P <0.05 vs. model group.(A higher resolution / colour version of this figure is available in the electronic copy of the article).

Fig. ( 8 )
Fig. (8).QF downregulated the protein levels of P-ERK1+2 and P-JNK and reduced mRNA levels of IL-8 in the lung tissues of RSVchallenged mice.(A) WB determination of P-ERK1+2 and P-JNK protein expression.(B-C) WB quantification of P-ERK1+2 and P-JNK.(D) mRNA expression of IL-8 was determined by real-time PCR.Data are presented as mean ± standard error.Significance: ## P <0.01 vs. control group; # P <0.05 vs. control group; ** P <0.01 vs. model group; * P <0.05 vs. model group.(A higher resolution / colour version of this figure is available in the electronic copy of the article).

Fig. ( 9
Fig. (9).Contd… Sun and Bin Yuan conceived and designed the research; Ya-Lei Sun, Pei-Pei Zhao, Cheng-Bi Zhu, and Xin-Min Li conducted the experiments and analyzed the data; Ya-Lei Sun and Pei-Pei Zhao wrote the paper; Bin Yuan reviewed and revised the manuscript.All authors have read and approved the final version of the manuscript.